1. Field of the Art
Generally, this application relates to molecular biology and microbiology chemistry processes and apparatuses including optical measuring or testing means. Certain embodiments relate to devices, systems, and methods for performing separations in an engineered material followed by matrix-assisted laser desorption/ionization (MALDI) in the same engineered material.
2. Background
There is a demand for miniaturization of traditional laboratory methods for analysis of biomolecules such as proteins and peptides. Advantages of miniaturization include smaller sample size, increased speed, and higher throughput. Currently, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) remains a ubiquitous sieving medium for protein separation and a foundational component of numerous commercially available analytical systems. Capillary electrophoresis (CE) has miniaturized the cross-sectional dimensions of SDS-PAGE but still requires long lengths to enable good separation resolution. Certain CE-based methods have enabled automation and accurate, repeatable quantification, but exhibit limited dynamic range and require a number of complex, time-consuming steps (see, e.g., O'Neill, et al. PNAS. 2006, 103: 16153-16158). Certain microfluidic-based methods have increased binding efficiency and reduced complexity, but these methods are also characterized by reduced separation resolution (see, e.g., Hughes, et al. PNAS. 2012, 109: 21450-21455).
Mass spectrometry (MS) techniques are commonly used for identification and quantification of proteins and peptides, as well as for analysis of post-translational modifications. MS is also used for analysis of other classes of biomolecules. Two of the most common ionization modes used in proteomics are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). For human cancer detection, including biomarker discovery and protein pattern signature identification, MALDI-MS is extensively used because of its high throughput and versatility. MALDI-MS has been successfully used for the analysis of many sample types including: fragile biomolecules, lipids, biopolymers, carbohydrates, and glycoconjugates. As a soft ionization technique (i.e., providing analyte ions with minimal fragmentation), MALDI-MS can be used for analysis of intact biomolecules and simple mixtures over a relatively broad mass range. MALDI-MS generally includes the use of specialized matrices, such as sinapinic acid, that promote ion formation. The matrix, however, also generates adducts (i.e., chemical addition product-caused artifacts) that can mask results, reduce mass accuracies, and preclude the analysis of analytes with low molecular weights. Co-crystallization of matrix and sample can also be problematic as heterogeneous co-crystallization or variations in crystal sizes can result in poor shot-to-shot reproducibility and reduced mass accuracy. The use of MALDI-MS in the field of glycomics, for example, is often hindered by poorly resolved peaks among differentially modified proteins, limitations in the ability to analyze small carbohydrates, and the appearance of matrix adducts that can mask results.
In view of the foregoing, what is needed in the art are new systems and methods for separating and analyzing mixtures of biological molecules.